In accordance with advances in physics, biochemistry, and bioinformatics, many technologies for obtaining profiles of a large number of biomolecules (proteins and carbohydrates) constituting a biosample have been developed. However, in spite of such technologies, there is a considerable need for novel and efficient methods and apparatuses due to problems regarding the use, maintenance cost, feasibility, accuracy, sensitivity, required testing times, and process automation ability of the existing methods or devices.
A method for generating comprehensive information on quantitative states of these biomolecules, that is, a profile of the biomolecules in the biosample containing the biomolecules, which is not an ultimate object but is a means for achieving the object, may be usefully used to identify microorganisms, cells, tissues, and the like, such that the method for generating a profile has been widely applied to medicine, veterinary medicine, environmental engineering, food engineering, agriculture, and the like.
A clinical decision support system (CDSS) for analyzing biological significance using the profile of the biomolecules in a biosample is a system for supporting a decision making process by a doctor, associated with diagnosis and treatment of a patient. The clinical decision support system is largely divided into a case-based machine learning inference system and an expert system. The case-based machine learning inference system is a system of collecting clinical information of patients with diagnosed diseases and biological information, that is, profile data on the biomolecules, and then inferring or determining a disease on the basis of given clinical information, biological information, and the like, using machine learning. The expert system is a system of diagnosing a disease using a rule predetermined by a medical expert.
A nucleic acid is a linear multimer in which nucleotides are linked by covalent bonds, wherein the nucleotide is a small organic compound composed of a phosphoric acid, a sugar, and a purine (adenine or guanine) or a pyrimidine (cytosine, thymine, or uracil). The nucleic acid exists in single-stranded form or double-stranded form, and a single-stranded nucleic acid forms a unique stereoscopic structure by hydrogen bonds and interactions between the nucleotides under specific physical conditions, and this stereoscopic structure is determined by a base sequence of the single strand.
In general, nucleic acids such as deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs) are information storage molecules for expression of proteins serving as components of cell structures or having activities such as enzymes, and the like. Since it was reported in 1982 that RNA can also have an enzyme activity by formation of a specific structure, many reports on structural characteristics of the nucleic acids and specific functions depending on the structural characteristics have been reported.
Nucleic acid consists of repeating units of four bases to form various stereoscopic structures with high diversity, and the nucleic acids form complexes through interaction with specific substances, such that this stereoscopic structure is stabilized.
Since nucleic acid may serve as a single-stranded nucleic acid (ligand) for molecules including proteins, nucleic acids binding to specific substances with high binding affinity and specificity are selected from a combinatorial library of single-stranded nucleic acids arranged in various base sequences through a predetermined selection process and through a base sequencing process.
A method for selecting a nucleic acid binding to a specific substance is referred to as systematic evolution of ligand by exponential enrichment (SELEX), and the selected nucleic acid is referred to as an aptamer (Tuerk C. and Gold L.; Science, 249, pp 505-510, 1990).
Nucleic acids (aptamers) capable of binding to various biomolecules, such as proteins capable of binding to nucleic acids in a natural state, or proteins that do not bind to the nucleic acids, have been selected through the SELEX as described above.
However, a method for selecting a nucleic acid through the existing SELEX is a method for selecting a nucleic acid (aptamer) having high binding affinity by preferentially mass-producing and purifying a corresponding protein (specific substance) prior to selection of a nucleic acid specifically binding to the specific substance (for example, the protein), reacting a library of single-stranded nucleic acids with the produced protein, and repeating selection and amplification. In this method, first, it was essential to secure the specific substance for selecting the nucleic acid. Therefore, in the method for selecting a nucleic acid through the SELEX according to the related art, a technical idea of selecting and using nucleic acids as a group having significance in a group of numerous unknown biomolecules contained in the biosample was not recognized. Biomolecule-single-stranded nucleic acid complexes may be selected by various methods. For example, a method for fixing the biomolecule to harvest a complex, and washing the complex, a capillary electrophoresis method, and the like, may be used.
Profiles of biomolecules including unknown molecules constituting tissues, cell mass, cells, microorganisms, and the like, which are biosamples, have been made by various methods using physical and chemical properties thereof. In general, a profile of biomolecules, which is a quantitative state of the biomolecules, in the biosample has been confirmed by performing electrophoresis on the biomolecules using molecular weights or pI values thereof.
In addition, a method for analyzing a profile to determine useful biomolecules, separating the useful biomolecules, and confirming constituent ingredients of the separated biomolecules using matrix assisted laser desorption/ionization-time spectroscopy of flight (MALDI-TOF), or the like, has been performed. Recently, various research into protein profiles has been conducted by surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOFMS) (Adam et al., Cancer Research, 62, 3609-3614. 2002; Li et al., Clinical Chemistry, 48, 1296-1304. 2002; and Petricoin et al., The Lancet, 359, 572-577. 2002).
Also recently, protein chips or aptamer chips have been developed and used by a high throughput screening method for proteomes (Smith et al., Mol. Cell Protomics, 11-18. 2003; and McCauley et al., Anal. Biochem., 319(2), 244-250. 2003). As a supporter used in the high throughput screening method as described above, there are a glass slide, a sensing surface of a biosensor, beads, nanoparticles, and the like.
Since arrangement of an antibody on a protein chip is known, the protein chip may be used to identify and quantify a specific substance. As a method for preparing a protein chip, there is a method of spotting an antibody to fix the antibody using a microarrayer. A protein chip sensing technology should be able to sense signals generated at very weak intensities in a state the protein chip is designed to integrate various antibodies at a high density on a small area in order to provide as much information as possible with one chip. Further, as bio-information of proteins is expanding, a degree of integration of the protein chip is also increasing, such that a novel method capable of rapidly and accurately quantitatively detecting a protein is required.
As a detection method, laser induced fluorescence spectroscopy has been mainly used up to now, and an electrochemical detection method, or the like, has been developed. A method for generating and analyzing a profile of specific proteins in a biosample using a protein chip has been developed through various processes as described above, but there are problems in that expensive apparatuses and reagents are used, and complicated procedures should be performed, etc., and there is a limitation in that the protein chip is applicable only to antigenic molecules.
An aptamer chip is different from the protein chip in that an aptamer (nucleic acid) is used instead of the antibody in the protein chip, and other factors of the aptamer chip are significantly similar to those of the protein chip.
As described above, methods for generating a quantitative state, that is, a profile, of biomolecules in a biosample using the protein chip and the aptamer chip have been developed, but there are problems in that expensive apparatuses and reagents are used, and complicated procedures should be performed, and the like. Particularly, there is a limitation in that protein chips or aptamer chips may be restrictively prepared only with respect to already known proteins from which antibodies or aptamers may be prepared.
Although a biosample is known to be composed of millions of proteins, only tens of thousands of proteins are identified. Therefore, there is a considerable need for a technology of generating a quantitative state, that is, a profile of unknown biomolecules such as unknown proteins in a biosample.
The present inventor suggested reverse-SELEX for generating a profile for proteome (see Korean Patent No. 10-0670799), an aptamer-based nucleic acid chip (see Korean Patent No. 10-0464225), a method for analyzing biological significance using an aptamer-based nucleic acid chip (see Korean Patent No. 10-0923048), and the like, but there were problems in that a suitable reference substance and a quality control method at the time of high throughput screening in order to generate proteome profiles were not suggested, and thus, it is impossible to perform suitable quality control upon generating and analyzing the profile. Therefore, the present invention was suggested in order to solve the problems as described above.
In research into a technology capable of entirely analyzing biomolecules in a biosample, analysis of profiles of biomolecules medically associated with diseases may be used to identify biomolecules that can serve as diagnostic markers, can be used to monitor therapeutic results, can play important roles in the outbreak or the progressions of diseases, are related to disease sensitivity, and can become target molecules for novel drug development.